Characterization of the putative ovarian stem cell marker DDX4 by mass spectrometry and fusion protein analysis of C-terminus expression

OBJECTIVE: Spermatogonial stem cell (SSC)-based technologies may have application for preserving and/or restoring male fertility. We evaluated the recovery of stem and progenitor spermatogonia from frozen/thawed testicular cell suspensions versus testicular tissue fragments frozen at a controlled rate (CR) or by vitrification. DESIGN: Laboratory study using human tissue. MATERIALS AND METHODS: Human testicular tissue was obtained through the Center for Organ Recovery and Education with approval from the University of Pittsburgh Institutional review board (#0506140). Testicular tissue was digested with enzymes to produce a cell suspension and frozen at CR or minced into large (6-10 mm3) or small (3-5 mm3) testicular tissue fragments and frozen at CR or by vitrification. The efficiency of each technique was analyzed by immunocytochemistry (ICC) for the spermatogonia marker UTF1 and human to nude mouse xenotransplantation. RESULTS: Cryopreservationmethodhad a significant effect on the recovery of UTF1 positive spermatogonia per gram of tissue (p 0.05). CONCLUSION: CR freezing of testicular tissue fragments (large or small) leads to the greatest recovery of UTF1 positive spermatogonia and transplantable SSCs. Recovery of SSCs from vitrified small testicular tissue fragments was also very good. Freezing and thawing of intact testicular tissue is compatible with several downstream applications, including testicular tissue grafting, testicular tissue organ culture or digestion with enzymes to produce a cell suspension for SSC transplantation. Supported by: This work was Supported by NIH grants HD055475 and HD061289, Magee-Womens Research Institute, Foundation and the Richard King Mellon Foundation and the United States-Israel Binational Science Foundation.